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Closing-wedge high tibial osteotomy: survival and risk factor analysis at long-term follow up
BMC Musculoskeletal Disorders volume 12, Article number: 46 (2011)
Closing-wedge high tibial osteotomy (HTO) is successful for the treatment of medial osteoarthritis with varus malalignment. Preoperative risk factors for HTO failure are still controversial. The aim of this study was to elucidate the outcome and assess the influence of risk factors on long term HTO survival.
199 patients were retrospectively studied with a mean follow-up period of 9.6 years after HTO. HTO failure was defined as the need for conversion to TKA. Survival was analyzed with the Kaplan-Meier method. Knee function was evaluated by the Hospital for Special Surgery (HSS) score. HTO-associated complications were also assessed. Univariate, multivariate, and logistic regression analysis were performed to evaluate the influence of age, gender, BMI, preoperative Kellgren-Lawrence osteoarthritis grade, and varus angle on HTO failure.
39 complications were recorded. Thus far, 36 HTOs were converted to TKA. The survival of HTO was 84% after 9.6 years. Knee function was considered excellent or good in 64% of patients. A significant preoperative risk factor for HTO failure was osteoarthritis, Kellgren-Lawrence grade >2.
HTO provides good clinical results in long-term follow-up. Preoperative osteoarthritis Kellgren-Lawrence grade >2 is a significant predictive risk factor for HTO failure. Results of HTO may be improved by careful patient selection. Complications associated with HTO should not be underestimated.
Closing wedge high tibial osteotomy (HTO) is an accepted procedure in the treatment of medial knee osteoarthritis with varus malalignment. Since Jackson and Waugh  initially described osteotomy below the tibial tubercle, various modifications of HTO have been published [2–7]. The fundamental goal of HTO is to partially offload the medial compartment and to realign the knee into valgus. Several studies have shown satisfactory short and medium term success; however, valgus-producing tibial osteotomy gradually deteriorates with time [8–10].
Ideal candidates for HTO are young and active patients who are not suitable for total knee arthroplasty (TKA) . Even though TKA is a popular and well-established method, younger age was associated with an increased risk of revision . Despite the fact that HTO results show its effectiveness, great debate on risk factors influencing HTO survival is still on-going. Various factors including age, gender, BMI, activity level, and varus angle are claimed to affect the duration of survival [13–18]. Selecting the ideal patient and identifying risk factors that may affect osteotomy longevity is important to obtain satisfactory results with HTO. The aim of this study was to determine HTO failure requiring conversion to TKA, including risk factors at long-term follow up. Furthermore, potential complications and clinical outcome of high tibial osteotomy were evaluated.
239 cases of closing-wedge osteotomy were performed between 1984 and 2001 at our institution. 13 (5.5%) patients were lost to follow-up. 27 (11%) patients were too ill or refused to participate in clinical and radiological assessment. With these patients, telephone interviews were performed to obtain information on further operations following HTO. 199 (83%) patients were therefore clinically and radiologically assessed. This study was approved by the ethics committee of the University Hospital Marburg and informed consent was obtained from all patients. This project was performed in accordance with the Helsinki Declaration, and with local legislation.
Patient demographics and clinical characteristics are illustrated in Table 1. The left knee was involved in 104 (52.2%) cases and the right in the remaining 95 (47.8%) cases. The average age at HTO was 54 ± 8 (25-72) years. The mean time of follow-up was 9.6 (1-18) years including 110 (55.3%) men and 89 (44.7%) women. A previous partial arthroscopic meniscectomy (less than one third) was performed in 51 (25.6%) cases.
The indication for HTO was symptomatic medial compartment osteoarthritis with varus malalignment. Preoperative radiographs included the full leg and standard short anteroposterior views in the standing position with the knee in full extension, as well as lateral and merchant views. The severity of osteoarthritis was scored according to the Kellgren-Lawrence system (0 normal, 4 severe ). Varus angle was measured using mechanical axes of the femur and the tibia (hip-knee-ankle angle) obtained on full length, full weight-bearing standing radiographs.
All patients were underwent a lateral closing-wedge technique . Fibular transection was performed at the junction of the middle and distal thirds  through a separate incision. A transverse incision with the patient in supine position was performed for the tibial osteotomy. The peroneal nerve was exposed and protected. The osteotomy was performed below the tibial tuberosity, leaving the medial cortex intact. Bone wedge size was based on the preoperative calculations from the long leg standing radiograph. A laterally-based wedge of bone was removed and the osteotomy was fixed with an AO-plate. Postoperative early active movement and physiotherapy were allowed. No casts were applied. Partial weight bearing using 2 crutches for 6 weeks was allowed until bony union was reached. Control standing X-rays were taken 6 weeks, 6 months, and 1 year after surgery as well as at time of latest follow-up.
The correlation between the conversion of HTO to TKA and possible risk factors including age, gender, body mass index (BMI), Kellgren-Lawrence grade of osteoarthritis, and varus angle were determined. Smoking was defined as a daily consumption of three or more cigarettes . All patients underwent physical examination and the application of a clinical score by co-author GA, an orthopaedic resident. Knee function was evaluated by the Hospital for Special Surgery (HSS) score , consisting of a questionnaire and physical examination. The items are clustered into six categories which include pain, function and range of motion, muscle strength, flexion deformity, and instability. A maximum score is 100 points. 85-100 points are excellent, 70-81 good, 60-69 fair, and <60 poor. Knee ROM was measured using a goniometer . An independent specialist in radiology evaluated the diagnostic images. All of the osteotomies were performed by two experienced knee surgeons. The operating surgeons were not involved in the clinical assessment.
HTO failure was defined as the need for conversion to TKA. Survival analysis was performed applying the Kaplan-Meier method. Potential risk factors (age, gender, BMI, Kellgren-Lawrence grade of osteoarthritis, and varus angle) were calculated by the univariate Log-Rank test. Cox regression models were used in both univariate (single risk factors) and multivariate (combinations of risk factors) analysis. Smoking as a risk factor for complications was calculated by Fisher's exact test. A p-value of < 0.05 was considered to be statistically significant.
At latest follow-up, 36 (16%) HTOs had been converted to TKA. The 5-year, 9.6 year, and 15-year HTO survival rates as determined by Kaplan-Meier analysis were 93%, 84%, and 68% (Figure 1). 54 patients had excellent results, 74 good, 51 fair and 20 poor. Pain was absent in 74 patients, mild in 66 cases, and severe in 58 cases (in motion but not at rest). No patient complained of continuous pain.
Partial arthroscopical meniscectomy prior to HTO did not influence HTO survival as shown by a multivariate Cox model (p = 0.65) and the log-rank test (p = 0.71).
Using the univariate and multivariate Cox regression model, age had no influence on HTO survival (p = 0.32 and p = 0.31, respectively). There was no significant difference between the survival rate of men and women using the multivariate Cox model (p = 0.32) and the Log-Rank test (p = 0.31). Univariate and multivariate Cox regression models found the preoperative varus angle not to be a significant factor (p = 0.8 and p = 0.84, respectively). The correlation between BMI and HTO failure was not significant using the univariate (p = 0.2) and multivariate (p = 0.11) Cox regression model as well as Log-Rank test (p = 0.45). Statistical significant correlation between the preoperative Kellgren-Lawrence osteoarthritis grade >2 and HTO failure could be detected with the use of both univariate (p = 0.003) and multivariate (p = 0.01) Cox regression models.
38 (19%) complications were recorded: 8 cases of deep vein thrombosis requiring low molecular weight heparin and compressive therapy; 1 vascular injury requiring surgery; 6 cases of peroneal palsy left with permanent deficit; and 3 superficial wound infections which responded to antibiotics. 9 patients had non-union of the osteotomized tibia and 8 of the fibula which required reostheosyntheses with bone grafting. Comparing obese and non-obese patients, no significant difference in complications was noted (p = 0.37). 6 of the 21 smokers had postoperative complications (3 non-union of the tibia, 2 non-union of the fibula, and 1 deep vein thrombosis). There was no significant difference in complications between smokers and non-smokers (p = 0.54).
We aimed to evaluate the clinical outcome of HTO and to assess potential risk factors that may influence its longevity. There were several limitations to our study, including its retrospective design and lack of control group. There were a significant number of patients who were lost to follow-up, although over 80% of patients could be included, which is satisfactory when compared with other studies in this area [18, 24].
Although there have been several studies on closing-wedge HTO, there is great variability in the results. Differences in patient outcome may be caused by wide heterogeneity among studies (e.g. different techniques and evaluation systems, varying degrees of deformity, and varus angle). Pooling the results is a challenge, as described by Amendola et al.  in a systematic review. There are few randomized controlled trials; well designed studies should include larger numbers to generate a higher quality of evidence. Long-term results are also needed for more solid conclusions. Thus, we conducted this study in order to select the ideal patient for HTO and improve the understanding of potential associated complications.
With the increasing numbers and success of TKA , fewer HTOs have been performed in the last several decades . Although Brouwer et al. failed to show in a systemic review that HTO was more effective than conservative treatment , it is an accepted method for medial gonarthritis with varus malalignment .
The complication rate of HTO is between 5.6% and 34% [30–32], including non-union and peroneal palsy. In our study, the 19% complication rate is within the range of published results . We performed all osteotomies below the tibial tuberosity, with 9 cases of non-union. Non-union of the tibia is an uncommon complication after closing-wedge HTO, but is more frequently found in osteotomies below rather above the tibial tubercle (14% vs 3.6%) . The risk of peroneal palsy is the most reported neurovascular complication after HTO . Wootton et al.  showed that the majority of peroneal damage occurred when fibular osteotomy is performed 8-15 cm below the fibular head - a zone that should be avoided when performing fibular osteotomy. Even when fibular transection was performed at the junction of the middle and distal thirds, 6 cases of peroneal palsy were noted. Peroneal palsy was a substantial contributing factor to patient dissatisfaction in our cohort.
Survival rate analysis revealed an 84% survival at 9.6 years follow-up, which is confirmed in other studies (75%-98%) [36–38]. Clinical outcome was also considered excellent or good in 64% cases 9.6 years postoperatively, similar to previous studies [31, 36, 39]. Results appear to gradually deteriorate after ten years [10, 13, 25].
Patient age is a crucial criterion for HTO indication. In our study, age was not a significant predictive factor for HTO failure. Contrary to our findings, Gstöttner et al.  and Trieb  demonstrated that age significantly influenced HTO survival.
As observed by other authors [11, 14, 37, 40], a significant association exists between preoperative Kellgren-Lawrence osteoarthritis grading and HTO failure. Patients with a higher grade of osteoarthritis (>2) may be better treated with arthroplasty. Huang et al.  reported that a large preoperative varus angle is influences HTO survival. We did not find this to be true in our cohort. A possible explanation for this discrepancy may be that in our patients, there was only a moderate average preoperative varus angle (6 degrees). However, two studies with similar preoperative varus angles (6°  and 6.5° ) confirmed our findings. Obesity is a growing problem in industrialized countries; Some studies have reported that HTO in obese patients is associated with early failure [15, 31, 42]. In our cohort, significant differences between obese and non-obese patients was not found . In a systematic review and meta-analysis, Blogojevic et al.  demonstrated female gender to be both a risk factor for osteoarthritis, and associated with severe OA. In our data, gender was not associated with HTO failure.
For several years, the lateral closing wedge HTO was the preferred treatment approach for patients with medial knee osteoarthritis. Due to some disadvantages of this technique, the medial opening wedge HTO regained popularity, since improvements in operative approach and special implants were developed [45, 46]. There is only one prospective, randomized study comparing lateral closing wedge and medial opening wedge osteotomies . At one year follow-up, both techniques reduced pain and improved knee function, but the difference was not significant.
A further option for treatment of unicompartimental gonarthrosis is unicompartimental arthroplasty (UKA). Even if there are differences in the indications for the two procedures, some patients can be treated either by HTO or UKA. Only a few studies compare the clinical outcome of UKA versus closing wedge HTO [48–50], and only two were randomized controlled studies [51, 52]. Data comparing UKA with HTO appear to support UKA over HTO. However, Brouwer et al.  stated in their meta-analysis that there is silver evidence for no significant difference in pain, knee function, and gait after HTO compared to UKA, and that HTO causes more complications than UKA.
HTO has a favorable outcome at 9.6 years of follow-up. A significant risk factor for HTO failure was preoperative Kellgren-Lawrence osteoarthritis grade >2. In order to improve the durability of HTO, appropriate patient selection is necessary. Surgeons should be aware that HTO is not without complications.
Jackson JP, Waugh W: Tibial osteotomy for osteoarthritis of the knee. J Bone Joint Surg Br. 1961, 43-B: 746-751.
Debeyre J, Artigou JM: [Long term results of 260 tibial osteotomies for frontal deviations of the knee]. Rev Chir Orthop Reparatrice Appar Mot. 1972, 58 (4): 335-339.
Coventry MB: Osteotomy about the knee for degenerative and rheumatoid arthritis. J Bone Joint Surg Am. 1973, 55 (1): 23-48.
Wagner H, Zeiler G, Baur W: [Indication, technic and results of supra- and infracondylar osteotomy in osteoarthrosis of the knee joint]. Orthopade. 1985, 14 (3): 172-192.
Devas MB: High tibial osteotomy for arthritis of the knee. A method specially suitable for the elderly. J Bone Joint Surg Br. 1969, 51 (1): 95-99.
Dietz FR, Weinstein SL: Spike osteotomy for angular deformities of the long bones in children. J Bone Joint Surg Am. 1988, 70 (6): 848-852.
Insall J, Shoji H, Mayer V: High tibial osteotomy. A five-year evaluation. J Bone Joint Surg Am. 1974, 56 (7): 1397-1405.
Gstottner M, Pedross F, Liebensteiner M, Bach C: Long-term outcome after high tibial osteotomy. Arch Orthop Trauma Surg. 2008, 128 (1): 111-115.
Koshino T, Yoshida T, Ara Y, Saito I, Saito T: Fifteen to twenty-eight years' follow-up results of high tibial valgus osteotomy for osteoarthritic knee. Knee. 2004, 11 (6): 439-444. 10.1016/j.knee.2004.03.005.
Virolainen P, Aro HT: High tibial osteotomy for the treatment of osteoarthritis of the knee: a review of the literature and a meta-analysis of follow-up studies. Arch Orthop Trauma Surg. 2004, 124 (4): 258-261. 10.1007/s00402-003-0545-5.
Ivarsson I, Myrnerts R, Gillquist J: High tibial osteotomy for medial osteoarthritis of the knee. A 5 to 7 and 11 year follow-up. J Bone Joint Surg Br. 1990, 72 (2): 238-244.
Robertsson O, Knutson K, Lewold S, Lidgren L: The Swedish Knee Arthroplasty Register 1975-1997: an update with special emphasis on 41,223 knees operated on in 1988-1997. Acta Orthop Scand. 2001, 72 (5): 503-513. 10.1080/000164701753532853.
Coventry MB, Ilstrup DM, Wallrichs SL: Proximal tibial osteotomy. A critical long-term study of eighty-seven cases. J Bone Joint Surg Am. 1993, 75 (2): 196-201.
Flecher X, Parratte S, Aubaniac JM, Argenson JN: A 12-28-year followup study of closing wedge high tibial osteotomy. Clin Orthop Relat Res. 2006, 452: 91-96. 10.1097/01.blo.0000229362.12244.f6.
Naudie D, Bourne RB, Rorabeck CH, Bourne TJ: The Install Award. Survivorship of the high tibial valgus osteotomy. A 10- to -22-year followup study. Clin Orthop Relat Res. 1999, 18-27. 367
Wolcott M: Osteotomies around the knee for the young athlete with osteoarthritis. Clin Sports Med. 2005, 24 (1): 153-161. 10.1016/j.csm.2004.08.002.
Matthews LS, Goldstein SA, Malvitz TA, Katz BP, Kaufer H: Proximal tibial osteotomy. Factors that influence the duration of satisfactory function. Clin Orthop Relat Res. 1988, 193-200. 229
Trieb K, Grohs J, Hanslik-Schnabel B, Stulnig T, Panotopoulos J, Wanivenhaus A: Age predicts outcome of high-tibial osteotomy. Knee Surg Sports Traumatol Arthrosc. 2006, 14 (2): 149-152. 10.1007/s00167-005-0638-5.
Kellgren JH, Lawrence JS: Radiological assessment of osteo-arthrosis. Ann Rheum Dis. 1957, 16 (4): 494-502. 10.1136/ard.16.4.494.
Aydogdu S, Yercan H, Saylam C, Sur H: Peroneal nerve dysfunction after high tibial osteotomy. An anatomical cadaver study. Acta Orthop Belg. 1996, 62 (3): 156-160.
Clemente Jiménez M, Pérez Trullén A, Rubio Aranda E, Marrón Tundidor R, Rodríguez Ibáñez M, Herrero Labarga I: A version of DSM-IV criteria adapted for adolescents and applied to young smokers. Arch Bronconeumol. 2003, 39 (7): 303-309.
Insall JN, Ranawat CS, Aglietti P, Shine J: A comparison of four models of total knee-replacement prostheses. J Bone Joint Surg Am. 1976, 58 (6): 754-765.
Watkins MA, Riddle DL, Lamb RL, Personius WJ: Reliability of goniometric measurements and visual estimates of knee range of motion obtained in a clinical setting. Phys Ther. 1991, 71 (2): 90-96. discussion 96-97
Gall N, Fickert S, Puhl W, Gunther KP, Stove J: [Predictors of tibial head transposition in the therapy of varus knee osteoarthritis]. Z Orthop Ihre Grenzgeb. 2005, 143 (5): 551-555. 10.1055/s-2005-872472.
Amendola A, Bonasia DE: Results of high tibial osteotomy: review of the literature. Int Orthop. 2010, 34 (2): 155-160. 10.1007/s00264-009-0889-8.
Kurtz SM, Lau E, Ong K, Zhao K, Kelly M, Bozic KJ: Future young patient demand for primary and revision joint replacement: national projections from 2010 to 2030. Clin Orthop Relat Res. 2009, 467 (10): 2606-2612. 10.1007/s11999-009-0834-6.
Wright JM, Crockett HC, Slawski DP, Madsen MW, Windsor RE: High tibial osteotomy. J Am Acad Orthop Surg. 2005, 13 (4): 279-289.
Brouwer RW, Raaij van TM, Bierma-Zeinstra SM, Verhagen AP, Jakma TS, Verhaar JA: Osteotomy for treating knee osteoarthritis. Cochrane Database Syst Rev. 2007, CD004019-3
Tunggal JA, Higgins GA, Waddell JP: Complications of closing wedge high tibial osteotomy. Int Orthop. 2010, 34 (2): 255-261. 10.1007/s00264-009-0819-9.
Wu LD, Hahne HJ, Hassenpflug T: A long-term follow-up study of high tibial osteotomy for medial compartment osteoarthrosis. Chin J Traumatol. 2004, 7 (6): 348-353.
Sprenger TR, Doerzbacher JF: Tibial osteotomy for the treatment of varus gonarthrosis. Survival and failure analysis to twenty-two years. J Bone Joint Surg Am. 2003, 85-A (3): 469-474.
Aglietti P, Buzzi R, Vena LM, Baldini A, Mondaini A: High tibial valgus osteotomy for medial gonarthrosis: a 10- to 21-year study. J Knee Surg. 2003, 16 (1): 21-26.
Vainionpaa S, Laike E, Kirves P, Tiusanen P: Tibial osteotomy for osteoarthritis of the knee. A five to ten-year follow-up study. J Bone Joint Surg Am. 1981, 63 (6): 938-946.
Aydogdu S, Cullu E, Arac N, Varolgunes N, Sur H: Prolonged peroneal nerve dysfunction after high tibial osteotomy: pre- and postoperative electrophysiological study. Knee Surg Sports Traumatol Arthrosc. 2000, 8 (5): 305-308. 10.1007/s001670000138.
Wootton JR, Ashworth MJ, MacLaren CA: Neurological complications of high tibial osteotomy--the fibular osteotomy as a causative factor: a clinical and anatomical study. Ann R Coll Surg Engl. 1995, 77 (1): 31-34.
Akizuki S, Shibakawa A, Takizawa T, Yamazaki I, Horiuchi H: The long-term outcome of high tibial osteotomy: a ten- to 20-year follow-up. J Bone Joint Surg Br. 2008, 90 (5): 592-596. 10.1302/0301-620X.90B5.20386.
van Raaij T, Reijman M, Brouwer RW, Jakma TS, Verhaar JN: Survival of closing-wedge high tibial osteotomy: good outcome in men with low-grade osteoarthritis after 10-16 years. Acta Orthop. 2008, 79 (2): 230-234. 10.1080/17453670710015021.
Papachristou G, Plessas S, Sourlas J, Levidiotis C, Chronopoulos E, Papachristou C: Deterioration of long-term results following high tibial osteotomy in patients under 60 years of age. Int Orthop. 2006, 30 (5): 403-408. 10.1007/s00264-006-0098-7.
Tang WC, Henderson IJ: High tibial osteotomy: long term survival analysis and patients' perspective. Knee. 2005, 12 (6): 410-413. 10.1016/j.knee.2005.03.006.
Aglietti P, Rinonapoli E, Stringa G, Taviani A: Tibial osteotomy for the varus osteoarthritic knee. Clin Orthop Relat Res. 1983, 239-251. 176
Huang TL, Tseng KF, Chen WM, Lin RM, Wu JJ, Chen TH: Preoperative tibiofemoral angle predicts survival of proximal tibia osteotomy. Clin Orthop Relat Res. 2005, 188-195. 10.1097/01.blo.0000149818.70975.07. 432
Spahn G, Muckley T, Kahl E, Hofmann GO: Biomechanical investigation of different internal fixations in medial opening-wedge high tibial osteotomy. Clin Biomech (Bristol, Avon). 2006, 21 (3): 272-278. 10.1016/j.clinbiomech.2005.10.010.
Amin AK, Clayton RA, Patton JT, Gaston M, Cook RE, Brenkel IJ: Total knee replacement in morbidly obese patients. Results of a prospective, matched study. J Bone Joint Surg Br. 2006, 88 (10): 1321-1326. 10.1302/0301-620X.88B10.17697.
Blagojevic M, Jinks C, Jeffery A, Jordan KP: Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis. Osteoarthritis Cartilage. 2010, 18 (1): 24-33. 10.1016/j.joca.2009.08.010.
Lobenhoffer P, Agneskirchner J, Zoch W: [Open valgus alignment osteotomy of the proximal tibia with fixation by medial plate fixator]. Orthopade. 2004, 33 (2): 153-160. 10.1007/s00132-003-0593-0.
Lobenhoffer P, Agneskirchner JD: Improvements in surgical technique of valgus high tibial osteotomy. Knee Surg Sports Traumatol Arthrosc. 2003, 11 (3): 132-138.
Brouwer RW, Bierma-Zeinstra SM, van Raaij TM, Verhaar JA: Osteotomy for medial compartment arthritis of the knee using a closing wedge or an opening wedge controlled by a Puddu plate. A one-year randomised, controlled study. J Bone Joint Surg Br. 2006, 88 (11): 1454-1459. 10.1302/0301-620X.88B11.17743.
Broughton NS, Newman JH, Baily RA: Unicompartmental replacement and high tibial osteotomy for osteoarthritis of the knee. A comparative study after 5-10 years' follow-up. J Bone Joint Surg Br. 1986, 68 (3): 447-452.
Ivarsson I, Gillquist J: Rehabilitation after high tibial osteotomy and unicompartmental arthroplasty. A comparative study. Clin Orthop Relat Res. 1991, 139-144. 266
Weale AE, Newman JH: Unicompartmental arthroplasty and high tibial osteotomy for osteoarthrosis of the knee. A comparative study with a 12- to 17-year follow-up period. Clin Orthop Relat Res. 1994, 134-137. 302
Borjesson M, Weidenhielm L, Mattsson E, Olsson E: Gait and clinical measurements in patients with knee osteoarthritis after surgery: a prospective 5-year follow-up study. Knee. 2005, 12 (2): 121-127. 10.1016/j.knee.2004.04.002.
Stukenborg-Colsman C, Wirth CJ, Lazovic D, Wefer A: High tibial osteotomy versus unicompartmental joint replacement in unicompartmental knee joint osteoarthritis: 7-10-year follow-up prospective randomised study. Knee. 2001, 8 (3): 187-194. 10.1016/S0968-0160(01)00097-7.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/46/prepub
We wish to thank Professor Michael Pfeiffer and Professor Peter Griss for performing the HTO procedure.
The authors declare that they have no competing interests.
TE drafted the manuscript and participated in its design and coordination; GA investigated, followed, and clinically managed patients; NT performed the statistical analysis; TJH, SFW, SL and BI participated in analysis and interpretation of data; MDS and UB initiated the study and participated in its design and coordination. All authors read and approved the final manuscript.
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